Toll-like receptor (TLR) proteins are known to play important roles in innate immunity via their capacity to recognize microbial products. TLR engagement by dendritic cells is necessary for the activation of these professional antigen presenting cells, which stimulates the secretion of pro-inflammatory and immunomodulatory cytokines. To date, much of our knowledge of TLR function in the immune system comes from the study of macrophages and dendritic cells. TLR proteins are also expressed on T lymphocytes, although their roles in the adaptive immune response remain unclear. Recent reports have shown that TLR2 can function as a co-stimulatory receptor for antigen-specific T cells. Moreover, CD4+CD45RO+ T memory cells (Tmem) constitutively express TLR2, and secrete IFN-gamma in response to activation by TLR2 agonists, but only when these cells are concomitantly stimulated by antigen, or by bystander cytokines. Thus, TLR2 may also participate in the generation and maintenance of immune memory. The relative contributions of TLR co-activation of dendritic cells and of T cells themselves to the development of Tmem cells remain unknown. This project will test the central hypothesis that TLR signaling is necessary to generate and maintain long-term memory in lymphoid and non-lymphoid tissues. These studies will provide key insights into how TLR agonists can be used as vaccine adjuvants that can generate long-lasting immune memory. The specific aims of this project will test two hypotheses. First, that engagement of TLR proteins on antigen-pulsed DCs can differentially prime for long-lived antigen-specific CD4+ Tmem cells in lymphoid and non-lymphoid tissues. Second, that engagement of TLR2 on the T cells themselves will differentially promote the generation of long-lived antigen-specific CD4+ Tmem cells in lymphoid and non-lymphoid tissues.